Project Summary The adaptive immune response to islet antigens, especially proinsulin, plays a central role in type 1 diabetes (T1D) development and pathology, with both T and B cells contributing to disease. Numerous large-scale prevention trials, mainly using preparations of insulin (subcutaneous, oral, and intranasal) to induce tolerance and delay the onset of clinical symptoms, have been unsuccessful. There exists the need to (1) study the autoimmune pathogenesis within the target organ of human T1D and (2) translate immunologic phenotypes from the islets to the blood of at risk subjects. We have recently identified proinsulin-reactive T cells from the islets of a T1D organ donor with a specific T cell receptor (TCR) responding to insulin B chain amino acids 9- 23 presented by HLA-DQ8 when transfected into a TCR null cell line. This T cell also responds to dispersed whole human islets, thus linking insulin B:9-23 T cell reactivity to human disease pathogenesis. Insulin B:9-23 specific CD4 T cells are present in the peripheral blood of new-onset T1D subjects. High affinity insulin- reactive B cells are found in the peripheral blood of healthy subjects, and these cells are restricted to the anergic B cell compartment (BND) and thus are antigen unresponsive. Importantly, in new-onset T1D patients and some first-degree relatives (FDRs) carrying high-risk HLA alleles, these B cells lose anergy. These findings lead us to hypothesize that insulin specific T and B cells undergo cognate interactions prior to the development of overt disease and concentrate in the pancreatic islets and lymph nodes where they propagate disease. This hypothesis will be tested in two specific aims. Utilizing the network for pancreatic organ donors (nPOD), Aim 1 focuses on defining antigen specificity, phenotypes and function of islet reactive immune cells from spleen and islets of organ donors with recent onset T1D. Aim 2 is a longitudinal study of at risk subjects with ?2 islet autoantibodies and HLA-DQ-DR matched autoantibody negative first degree relatives from the TrialNet Pathway to Prevention (PTP) Living Biobank (previously referred to as the Natural History Study) measuring functional insulin specific T cell responses, enumerating insulin binding B cells, and characterizing insulin autoantibodies for antigen affinity and subtypes. The proposed studies will advance our long-term goal of bridging the gap of knowledge between the target organ and blood for biomarkers of disease progression and ultimately T1D prevention with enhanced understanding of disease pathogenesis.